This invention is in the general field of biochemical engineering. More specifically, the subject invention relates to the preparation of biologically active recombinant lipophilic proteins such as human interferons and interleukin-2. Still more specifically, the subject invention relates to an improved process for the production and recovery of lipophilic proteins from genetically transformed host organisms, lipophilic protein preparations relatively high purity, and therapeutically acceptable formulations thereof.
Interleukin-2 (IL-2) is a lymphokine which is produced by normal peripheral blood lymphocytes and induces proliferation of antigen or mitogen stimulated T cells after exposure to plant lectins, antigens, or other stimuli. IL-2 was first described by Morgan, D. A., et al., Science (1976) 198: 1007-1008 and was then designated T cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes. Now renamed as interleukin-2, IL-2 later has been found also to modulate a variety of functions of immune system cells in vitro and in vivo. IL-2 is one of several lymphocyte-produced messenger-regulatory molecules which mediate immunocyte interactions and functions.
Human IL-2 has been obtained from genetically engineered E. coli as an unglycosylated protein with biological activities equivalent to those of native, glycosylated IL-2. [Devos et al., Nuc. Acids. Res., 11: 4307-4323 (1983); Rosenberg et al., Science, 223: 1412-1415 (1984); Wang et al., Science, 224: 1431-1433 (1984); and Doyle et al., J. Biol. Resp. Modifiers, 4: 96-109 (1985)] Rosenberg and his co-workers have shown that systemic administration of recombinant IL-2 in high doses causes regression of established metastases in mice [Rosenberg et al., J. Exp. Med., 161: 1169-1188 (1985)]; and, in conjunction with lymphokine-activated killer cells, in humans [Rosenberg et al., New Eng. J. Med., 313: 1485-1492 (1985)].
IL-2 was initially made by cultivating human peripheral blood lymphocytes (PBL) or other IL-2-producing cell lines. See, for example, U.S. Pat. No. 4,401,756. Recombinant DNA technology has provided an alternative to PBLs and cell lines for producing IL-2. Taniguchi, T., et al., Nature (1983) 302: 305-310 and Devos, R., Nucleic Acids Research (1983) 11: 4307-4323 have reported cloning the human IL-2 gene and expressing it in microorganisms.
Naturally occurring interferons (IFNs) are species-specific proteins, often glycoproteins, produced by various cells upon induction with viruses, double stranded RNAs, other polynucleotides, antigens and mitogens. Interferons exhibit multiple biological activities such as antiviral, antiproliferative, immunomodulatory and anticellular functions. At least three distinct types of human interferons have been identified and characterized in terms of their anti-viral, anti-growth and activation of natural killer cell (NK) activities. They are produced by leukocytes, lymphocytes, fibroblasts and the immune system and are classified as .alpha., .beta. and .gamma. interferons. These are reported to be different proteins coded for by distinct structural genes.
Native human .beta.-interferon (.beta.-HIFN) is generally produced by superinducing human fibroblast cultures with poly-IC (polyriboinosinic acid and polyribocytidylic acid) and isolating and purifying the .beta.-HIFN thus produced by chromatographic and electrophoretic techniques. Proteins or polypeptides which exhibit native .beta.-interferon like properties may also be produced using recombinant DNA technology by extracting poly-A-rich 12S messenger RNA from virally induced human cells, synthesizing double-stranded c-DNA using the m-RNA as a template, introducing the c-DNA into an appropriate cloning vector, transforming suitable microorganisms with the vector, harvesting the bacteria and extracting the .beta.-HIFN therefrom. Nagola, S. et al., Nature, 284: 316 (1980); Goeddel, D. V. et al., Nature, 287: 411 (1980); Yelverton, E. et al., Nuc. Acid Res., 9: 731 (1981); Streuli, M. et al., Proc. Nat'l. Acad. Sci. (U.S.), 78: 2848 (1981); European Pat. Application Numbers 28033, published May 6, 1981; 321134, published Jul. 15, 1981; 34307 published Aug. 26, 1981; and Belgian Patent 837397, issued Jun. 1, 1981 describe various currently used methods for the production of .beta.-interferon employing recombinant DNA techniques. The expressed proteins or polypeptides have been purified and tested and have been found to exhibit properties similar to those of native IFNs. Bacterially produced IFNs thus appear to have potential therapeutic use as antiviral and antitumor agents and the production of IFNs by such bacterial fermentations is expected to yield sufficiently large quantities of IFN at a relatively low cost for clinical testing.
Protein samples for use in clinical studies, however, must be of relatively high purity and substantially uncontaminated with toxic host cell constituents, cell debris and other extraneous chemicals introduced during the extraction and purification steps. There are several methods currently available for the preparation, recovery and purification of bacterially produced proteins.
U.S. Pat. No. 4,315,852 to Leibowitz et al. describes and claims a method for the acid extraction of leukocyte interferon from bacterial cells and neutralization of the extractant to obtain the interferon.
Derynck et al., Nature, 287: 193 (1980) teach lysing transformed E. coli cells using a solution containing 5M urea, 1% sodium dodecyl sulfate (SDS), and 1% 2-mercaptoethanol. The lysate, which was purified by chromatography, exhibited interferon activity.
Scandella and Kornberg, Biochemistry, 10: 4447 (1971) describe the preparation of a phospholipase from E. coli by solubilizing the cell membranes with SDS and precipitating the solubilized protein with 1-butanol.
U.S. Pat. No. 4,343,735, to Menge et al. teaches a process for the purification of interferon by partitioning it in an aqueous multi-phase system in the presence of ion exchangers which are soluble in the system and are derivatives of polyethers.
U.S. Pat. No. 4,343,736 to Uemura et al. discloses a method for recovering interferon by absorption on water-insolubilized heparin and then eluting the interferon with an aqueous solution of an inorganic salt and chondroitin sulfate.
U.S. Pat. No. 4,289,689 to Friesen et al. discloses how to recover and purify human native .beta.-interferon by use of affinity chromatography and high pressure liquid chromatography.
U.S. Pat. No. 4,460,574 to Yabrov discloses a pharmaceutical composition comprising native human .alpha.- and .beta.-interferons used for rectal or urogenital treatment of human interferon-sensitive diseases.
U.S. Pat. No. 4,364,863 to Leibowitz et al. describes a method of extracting fibroblast interferon from bacteria using a low pH followed by a high pH extraction procedure.
PCT WO 80/02229 to Benzon discloses purification of alpha (leukocyte) interferon, which is not a lipophilic protein.
EP 42,246 discloses that recombinant interferons may be dissolved in any pharmaceutically acceptable non-toxic carrier appropriate for the desired form of administration without further details.
U.S. Pat. No. 4,450,103 discloses solubilizing the protein in an aqueous medium with an appropriate solubilizing agent, extracting the protein from the aqueous medium with 2-butanol or 2-methyl-2-butanol, and precipitating the protein from the alcohol phase.
Cancer Treatment Reports, 62, 1900-1906 (1978) and EP 89,245 disclose that native beta-interferon may be formulated directly with human serum albumin in a pharmaceutically compatible aqueous-based medium at a pH of 7.2-7.8.
Copending U.S. Ser. No. 780,551, filed Sep. 26, 1985, entitled "Stable Formulation of Biologically Active Proteins for Parenteral Injection" describes a pharmaceutical composition suitable for parenteral injection into humans or animals containing IL-2 or IFN-.beta. dissolved in a suitable carrier medium at pH 7.0 to 8.0 stabilized with sodium laurate. The formulation can be prepared by adding to either protein, after its recovery from a transformed organism, an effective amount of sodium laurate at a pH of 9 to 9.5 and then adjusting the pH of the formulation to between 7.0 and 8.0.
EP 158,487, published Oct. 15, 1985, discloses an interleukin-2 composition which comprises human serum albumin, a reducing compound or a combination thereof, and which is adjusted to a pH from 3 to 6 as a solution.
Alpha-interferons and native beta-interferon are not lipophilic proteins. Therefore, they can be stabilized and solubilized by adding a stabilizer such -as human serum albumin directly to the formulation at physiological pH. In contrast, lipophilic proteins such as recombinant beta-interferon and interleukin-2 are not solubilized by addition of human serum albumin at pH 6.8-7.8.
A major problem with the existing methods of purification and recovery of lipophilic proteins is that the protein is not produced in a sufficiently pure form and in sufficiently large quantities for clinical and therapeutic purposes. The resulting protein preparations, especially those that are produced by recombinant DNA techniques, also have residual amounts of chemicals, such as SDS and other surfactants or precipitants used in the extraction and purification steps. It would be desirable, therefore, to have available a process for the recovery of a lipophilic protein in sufficiently large quantities and sufficiently pure form for clinical and therapeutic applications. Also, it would be desirable to have formulations substantially free of residual chemicals or which have very low levels of such residual chemicals.
Accordingly, it is an object of the present invention to provide a pharmaceutically acceptable sample of a lipophilic protein such as recombinant .beta.-interferon or interleukin-2 which is of relatively high purity and in sufficiently large quantities for clinical and therapeutic applications.
Yet another object of the instant invention is to provide lipophilic proteins such as recombinant .beta.-interferon or interleukin-2 preparations which are substantially free of SDS or contain very low levels of SDS without loss of the lipophilic protein's biological activity.
A further object of this invention is to provide recombinant .beta.-interferon and interleukin-2 samples wherein the level of SDS is less than about 10 p.p.m.
U.S. Pat. No. 4,462,940 which is in the series of patents and applications of which this application is a continuation-in-part, describes an improved method for the production, recovery and purification of a lipophilic protein such as human recombinant .beta.-interferon and interleukin-2 and comprises solubilizing the protein into an aqueous medium with a suitable solubilizing agent, extracting the solubilized protein with an aliphatic alcohol, precipitating the protein from the alcohol phase with an aqueous buffer, and diafiltering the protein at a pH of about 10.5 to 12.5, preferably at a pH of about 12, against water adjusted to a pH of about 10.5 to 12.5, preferably about 12, or against mixtures of water and aliphatic alcohols, preferably ethanol and glycerol adjusted to a pH of about 10.5 to 12.5, preferably about 12, substantially to remove SDS or to reduce its concentration. The protein sample is optionally purified by conventional methods such as chromatography prior to the diafiltration.
A preferred embodiment of the above method comprises recovering bacterially produced human .beta.-interferon by disruption of the bacterial cells, solubilization of the interferon with a suitable solubilizing agent, extracting the solubilized interferon with an aliphatic alcohol of 2-6, preferably 4-6 carbon chain length, precipitating the interferon from the alcohol phase, further purifying the interferon by conventional methods, preferably gel filtration chromatography, and diafiltering the interferon fraction at a pH of about 10.5 to 12.5, preferably at a pH of about 11, against pure water or mixtures of water and aliphatic alcohols, preferably methanol, ethanol, propanol, butanol, glycerol and the like, also adjusted to a pH of about 10.5 to 12.5, preferably about 11. Said process results in human .beta.-interferon formulations with levels of SDS less than about 10 p.p.M.